PROJECT SUMMARY In situ immunofluorescence imaging is a powerful method to study the locations, expression levels and structures of proteins in cells and tissues. In particular, multiplexed imaging reveals the interaction networks of proteins, which allows us to understand the underlying mechanisms of many diseases. However, it has been challenging to perform multiplexed immunofluorescence imaging due to its extremely time-consuming process, high cost and lack of signal amplification. The limited spatial resolution achievable with confocal microscopy often fails to reveal complex spatial organization and to determine localizations of proteins. Here we propose super-multiplexed immunofluorescence nanoscopy that is capable of imaging more than twenty different proteins in 24 hours with nanoscale resolution. We will employ DNA-barcoded secondary nanobodies that are monovalent, open-source and designed for quantitative labeling. Repeated introduction and washing of fluorescent DNA imagers will generate highly multiplexed images. Moreover, we will develop unprecedentedly fast stimulated emission depletion (STED) microscopy that employs a parallelized line array of doughnut beams. It will feature a large imaging area and excellent optical sectioning capability. Photon reassignment, hyperspectral imaging and deep-learning will further facilitate rapid super-resolution-based protein profiling. Our new biochemical and optical tools will play crucial roles in diverse biomedical areas including brain proteomics and cancer profiling.